Fluorescent entity comprising a fluorophore covalently attached to at least one oligonucleotide and comprising at least one functional group, and uses thereof

ABSTRACT

The invention relates to a fluorescent entity comprising a fluorophore, with the exception of a rare earth metal cryptate, covalently attached to one or more oligonucleotide(s) or oligonucleotide analog(s) and comprising at least one functional group, introduced or generated on the fluorophore or one of the oligonucleotides or oligonucleotide analogs, allowing it to be coupled with a carrier molecule. The invention also relates to a fluorescent conjugate consisting of a fluorescent entity covalently attached to a carrier molecule.

The invention relates to a fluorescent entity comprising a fluorophore,covalently attached to one or more oligo-nucleotides or oligonucleotideanalogs and comprising at least one functional group, introduced orgenerated on the fluorophore or one of the oligonucleotides oroligonucleotide analogs.

Many families of organic molecules are used as fluorescent labels forbiomolecules in a number of applications, in particular for diagnosticmethods which make it possible to follow or to quantify thesebiomolecules.

Mention may in particular be made of rhodamines, cyanins, squaraines orbodipy dyes. Most of these molecules have a high molar extinctioncoefficient (often greater than 100 000 M¹ cm¹) and a quantum yield offluorescence generally greater than 20%.

However, due to their often very hydrophobic nature, these organicmolecules have a tendency to form aggregates at the surface ofbiomolecules, in particular proteins, when the desire is to have severalfluorescent labels per protein (U. Schobel et al., Bioconjugate chem.,1999, 10, 1107-1114). Since these aggregates are virtuallynonfluorescent, the mean quantum yield of the fluorescent moleculespresent at the surface of the proteins is then significantly lower thanthat of the native molecules.

To overcome this problem, an attempt has been made, in the literature,to increase the hydrophilic nature of the fluorescent molecules, inparticular by adding sulfonate groups (U.S. Pat. No. 5,268,486). Mentionmay also be made of the addition of sugars or of carbohydrate residuesto the structure of the fluorescent molecules (WO 98/49176). However,the addition of these units limits the aggregation phenomenon withoutsuppressing it.

Other approaches have also been used to avoid the decrease in quantumyield of fluorescence after labeling of proteins. Application WO98/26287 describes a method using cyclodextrins to encapsulatefluorescent molecules at the surface of proteins and to limit theiraggregation. However, this technique does not function satisfactorilywith all types of molecules.

A technique has recently been described which makes it possible toobtain proteins having, at their surface, only a single fluorescentmolecule and makes it possible to thus be able to maintain a highquantum yield by avoiding the aggregation phenomenon (Winckler et al.,Specific labeling of proteins using reactive affinity tag-dye systems,SBS Conference, Vancouver, 2000). This system is limiting since it doesnot make it possible to have several fluorescent molecules per protein.

The problem to be solved therefore consists in providing a label whichcan be attached to a biomolecule, the photophysical properties of whichare not modified, in particular by aggregation phenomena, when severalof these labels are simultaneously attached to a biomolecule, inparticular a protein.

According to the invention, this problem can be solved by attaching thefluorophore to one or more oligo-nucleotide(s) or oligonucleotideanalog(s), the compound thus formed also comprising one or morereactional group(s) allowing it to be attached to a carrier molecule.

According to a first aspect, the invention therefore relates to afluorescent entity comprising a fluorophore, with the exception of arare earth metal cryptate, covalently attached to one or moreoligonucleotide(s) or oligonucleotide analog(s), characterized in thatit comprises at least one functional group, introduced or generated onthe fluorophore or on one of the oligo-nucleotides or oligonucleotideanalogs.

The fluorophore of the entity according to the invention preferablycomprises one or more aromatic rings and has a high molecular extinctioncoefficient, greater than 20 000, preferably greater than 50 000.

Said fluorophore is preferably chosen from rhodamines, cyanins,squaraines, bodipys, fluoresceines and their derivatives.

The term “oligonucleotide” denotes equally oligodeoxyribonucleotides(DNA fragment) or oligoribonucleotides (RNA fragment).

The term “oligonucleotide or oligonucleotide analog” is intended tomean, in the present description:

-   -   either a series of ribonucleotide or deoxyribonucleotide units        attached to one another via bonds of the phosphodiester type;    -   or a series of ribonucleotide or deoxyribonucleotide units or of        nucleotide analog units modified on the sugar or on the base and        attached to one another by natural internucleotide bonds of the        phosphodiester type, some of the internucleotide bonds being        optionally replaced with phosphonate, phosphoramide or        phosphorothioate bonds. These various oligo-nucleotide families        are described in Goodchild, Bioconjugate Chemistry, 1(3),        May/June 1990, 77-99;    -   or a series comprising both ribonucleotide or        deoxyribonucleotide units attached to one another by bonds of        the phosphodiester type and nucleoside analog units attached to        one another by amide bonds, commonly called “PNAs” (“peptide        nucleic acids”), as described in M. Egholm et al., J. Am. Chem.        Soc., 1992, 114, 1895-1897; such compounds are, for example,        described in R. Vinayak et al., Nucleoside & Nucleotide, 1997,        16 (7-9), 1653-1656;    -   or a series of ribonucleotide or deoxyribonucleotide units in        which some of the nucleosides or of the internucleotide bonds        have been modified compared to a natural oligoribonucleotide or        oligodeoxyribonucleotide formed from the common nucleosides        (adenosine, deoxyadenosine, cytidine, deoxycytidine, guanosine,        deoxyguanosine, uridine or thymidine) attached by phosphate        bridges, for example a phosphorothioate oligonucleotide (OPT) in        which all or part of the phosphate bridges have been replaced        with thiophosphate bridges (P. J. Romaniuk, F.        Eckstein (1982) J. Biol. Chem. 257, 7684).

The use of each one of these types of oligonucleotide constitutes anadvantageous aspect of the invention.

The term nucleotide or nucleoside “analog” is intended to mean anucleotide/nucleoside comprising at least one modification relating tothe sugar or the nucleobase or a combination of these modifications. Byway of example, mention may be made of the following modifications:

I. Modifications Concerning the Sugar (Nucleotide or NucleosideAnalogs):

1°) The sugar component can be modified in that the configuration of thehydroxyls (free or involved in a phosphate bridge) is different from thenatural configuration (which is, respectively, β-D-erythro in the DNAseries and β-D-ribo in the RNA series), as in the analogs having thebackbone β-D-arabino-pentofuranoside or β-D-xylo-pentofuranoside, forexample.

20°) The structure can be modified in that the internucleotide bonds areof the 2′→5′ type, such as in the case ofβ-D-ribo-pentofuranoside-2′-phosphate or3′-deoxy-β-D-erythro-pentofuranoside-2′-phosphate derivatives.

Nucleotides exist in which the structure includes the preceding twomodifications, such as β-D-xylo-pentofuranoside-2′-phosphate.

3°) The structure can differ from the natural model in that the 4′carbon has the opposite configuration, this is the caseα-L-threo-pentofuranoside-3′-phosphate. The difference may relate to theconfiguration of the carbon in the 1′-position (anomeric position), thisis the case of α-D-erythro-pentofuranoside-3′-phosphate.Nucleotides/nucleosides exist in which the structure includes thepreceding two modifications, such asβ-L-threo-pentofuranoside-3′-phosphate.

4°) The structure can differ from the natural model in that the oxygenin the 4′-position is replaced with a carbon (carbocyclic analog) orwith a sulfur, such as 4′-thio-β-D-erythro-pentafuranoside-3′-phosphate.

5°) The structure can differ from the natural model in that one of thehydroxyls of the sugar is alkylated, for example in the backbone2′-O-alkyl-β-D-ribo-pentafuranoside-3′-phosphate, the alkyl grouppossibly being, for example, the methyl or allyl group.

6°) The structure can differ from the natural model in that only thesugar component is conserved, such as in1,2-dideoxy-D-erythro-pentafuranose-3-phosphate, or in that the sugar isreplaced with a polyol such as propanediol.

II. Modifications Concerning the Nucleobase (Nucleotide Analogs):

1°) The nucleobase can be modified in that the substituents of thenatural bases are modified, such as in 2,6-diaminopurine, hypoxanthine,4-thiothymine, 4-thiouracil or 5-ethynyluracil.

2°) The positions of the substituents can be switched compared to thenatural bases, such as in isoguanosine or isocytosine.

3°) A nitrogen atom of the nucleobase can be replaced with a carbon, asin the 7-deazaguanosine or 7-deazaadenine.

III. Modifications Concerning the Internucleotide Bond:

Moreover, as mentioned above, the bonds between the sugar units or theiranalogs can also be modified, for example by replacing one or more ofthe oxygen atoms of the natural phosphodiester bond with a carbon(phosphonate series), a nitrogen (phosphoramide series) or a sulfur(phosphorothioates).

The internucleotide bonds can also be replaced with amide bonds, as inoligonucleotide analogs of the “PNA” type.

According to a preferred aspect, the oligonucleotide consists of aseries comprising both ribonucleotide or deoxyribonucleotide unitsattached to one another by bonds of the phosphodiester type andnucleoside analog units attached to one another by amide bonds.

In particular, in this case, said oligonucleotide can comprise at leastfive internucleotide bonds of the phosphodiester type at the endintended to be attached to the fluorophore.

Preferably, said oligonucleotide or oligonucleotide analog comprisesfrom 5 to 60 nucleotide units, in particular from 5 to 20, preferably 5to 15 nucleotide units.

The fluorescent entity according to the invention should comprise atleast one functional group which allows it to be coupled with a carriermolecule.

Advantageously, the functional group is an amine function of anucleotide unit of the oligonucleotide or of the oligonucleotide analog,or results from the reaction of a free amine function of a nucleotideunit of the oligonucleotide or of the oligonucleotide analog using ahomobifunctional or heterobifunctional reagent which makes it possibleto introduce a functional group chosen from the groups: activated esterof a carboxylic acid, carboxylic acid, isothiocyanate, aldehyde,carbonyl, sulfonyl halide, alkyl halide, azide, hydrazide,dichlorotriazine, anhydride, haloacetamide, maleimide and sulfhydryl.The homobifunctional and heterobifunctional reagents and also their useare described in “Bioconjugation” (chapters 5.3 to 5.6, M. Aslam & A.Dent, Macmillan, London, 1998).

Said functional group can, for example, result from the reaction of afree amine function of a nucleotide unit of the oligonucleotide or ofthe oligonucleotide analog, with an N-hydroxysuccinimidyl ester.

According to a preferred aspect, the functional group is chosen from thegroups: maleimide, carboxylic acid, haloacetamide, alkyl halide, azido,hydrazido, aldehyde, ketone, amino, sulfhydryl, isothiocyanate,isocyanate, monochlorotriazine, dichlorotriazine, aziridine, sulfonylhalide, acid halide, hydroxysuccinimide ester, hydroxy-sulfosuccinimideester, imido ester, hydrazide, azido-nitrophenyl, azidophenyl, azide,3-(2-pyridyldithio)-proprionamide and glyoxal, and more particularly thegroups of formula:

where n ranges from 0 to 8 and p is equal to 0 or 1, and Ar is a 5- or6-membered heterocycle comprising 1 to 3 hetero atoms, optionallysubstituted with a halogen atom.

According to an advantageous aspect, the functional group(s) is (are)attached to the fluorophore and/or to the oligonucleotide by a spacerarm consisting of a divalent organic radical, chosen from linear orbranched C₁-C₂₀ alkylene groups optionally containing one or more doublebonds or triple bonds and/or optionally containing one or more heteroatoms, such as oxygen, nitrogen, sulfur, phosphorus, or one or morecarbamoyl or carboxamido group(s); C₅-C₈ cycloalkylene groups and C₆-C₁₄arylene groups, said alkylene, cycloalkylene or arylene groups beingoptionally substituted with alkyl, aryl or sulfonate groups.

In particular, the spacer arm is chosen from the groups:

in which n₁ and n₂ are between 2 and 6.

According to a preferred aspect, the invention relates to a fluorescententity of formula (I)

in which:

-   -   A represents a group chosen from:         —N(R₃)_(r)    -   r=2 or 0.3    -   the dashed lines each represent the carbon atoms required to        form 1 to 3 fused rings, the groups R₃ being attached to these        rings;    -   X and Y each represent N, C═O, O, S or C(CH₃)₂    -   m has a value 1, 2, 3 or 4;    -   q has a value 1, 2 or 3;    -   (R₃)_(q) represents q groups R₃, which may be identical or        different;    -   the groups R₁, R₂ and R₃ are identical or different and are        chosen from hydrogen; a group —(CH₂)_(s)-Z in which s ranges        from 0 to 4 and Z represents a group CH₃, SO₃H, OH or N⁺R₁R₂R₃        in which R₁, R₂ and R₃ are as defined above; a functional group        as defined above; and an oligonucleotide or oligonucleotide        analog optionally comprising a functional group as defined        above;    -   R₄ is chosen from: H; OH; CH₃; Cl and the groups of formula:    -   the substituents R₄ in the allylic position possibly forming,        with the polyethylenic chain, 1 to 3 fused rings containing from        4 to 14 atoms, which may or may not be saturated, said rings        possibly containing one or more atoms of O, N and S, and        possibly being optionally substituted with an oxo group.

Preferred fluorescent entities according to the invention correspond toformulae (II) and (III)

in which the dashed lines, R₁, R₂, R₃, R₄, X, m and q are as definedabove for formula (I).

Advantageously, the invention relates to fluorescent entities of formula(IV), (V), (VI) or (VII)

in which R₁, R₂, R₃ and R₅ are identical or different and are chosenfrom hydrogen; a group —(CH₂)_(s)-Z in which s ranges from 0 to 4 and Zrepresents a group CH₃, SO₃H, OH or N⁺R₁R₂R₃ in which R₁, R₂ and R₃ areas defined above; a functional group or an oligonucleotide oroligonucleotide analog as defined above.

According to another preferred aspect, the invention relates to afluorescent entity of formula (VIII)

in which the substituents R₆ to R₁₂ are chosen from: hydrogen; ahalogen; an alkyl; a cycloalkyl; aryl; arylalkyl; acyl; sulfo; afunctional group or an oligonucleotide or oligonucleotide analog asdefined above.

Another fluorescent entity according to the invention corresponds toformula (IX)

in which R₁, R₂, R₃, R₄, X, Y, m and q are as defined above.

Entities of formula (IX) which are particularly preferred are those inwhich X and Y represent a group C(CH₃)₂, and also those in which

-   -   R₁ and R₂ represent an alkyl comprising from 1 to 4 carbon atoms        or a group of formula below, at least one of the groups R₁ and        R₂ representing a group of formula below:    -   R₄ represents hydrogen    -   q=1, m=2

R₃ represents hydrogen; a group —(CH₂)_(s)-Z in which s ranges from 0 to4 and Z represents a group CH₃, SO₃H, OH or N⁺R₁R₂R₃ in which R₁, R₂ andR₃ are as defined above; a functional group or an oligonucleotide oroligonucleotide analog as defined above;

-   -   R₄ is chosen from: H; OH; CH₃; Cl and the groups of formula:        the substituents R₄ in the allylic position possibly forming,        with the polyethylenic chain, 1 to 3 fused rings containing from        4 to 14 atoms, which may or may not be saturated, said rings        possibly containing one or more atoms of O, N and S, and        possibly being optionally substituted with an oxo group.

Advantageously, the fluorescent entity according to the inventioncomprises a fluorophore which is covalently attached to theoligonucleotide, either directly or via a spacer arm.

This spacer arm may, for example, consist of a divalent organic radicalchosen from linear or branched C₁-C₂₀ alkylene groups optionallycontaining one or more double bonds or triple bonds and/or optionallycontaining one or more hetero atoms, such as oxygen, nitrogen, sulfur,phosphorus, or one or more carbamoyl or carboxamido group(s); C₅-C₈cycloalkylene groups and C₆-C₁₄ arylene groups, said alkylene,cycloalkylene or arylene groups optionally being substituted with alkyl,aryl or sulfonate groups, or chosen from the groups:

in which n₁ and n₂ are between 2 and 6.

According to a subsequent aspect, the invention also relates to thefluorescent conjugates consisting of an entity as defined abovecovalently attached to a carrier molecule.

Advantageous conjugates are those in which the final molar ratio,defined as the number of moles of fluorescent entities per carriermolecule, is greater than 0 and less than 100, preferably less than 20.

The carrier molecule is, for example, an antibody, an antigen, anintracellular messenger, an intercellular messenger, a protein, apeptide, a hapten, a lectin, biotin, avidin, streptavidin, a toxin, acarbohydrate, an oligosaccharide, a polysaccharide, a nucleic acid, ahormone, a vitamin, a medicinal product, a polymer, a polymericparticle, glass, a particle of glass or a surface made of glass or of apolymer.

The use of the fluorescent entities according to the invention makes itpossible to produce conjugates exhibiting virtually zero aggregation ofthe fluorophore. Consequently, the quantum yield of the fluorophore canbe almost completely conserved after attachment to carrier molecules,even when the final molar yield (number of moles of fluorescent entitiesper carrier molecule) increases.

This makes these conjugates very advantageous for use in a fluorescentsystem using nonradiative energy transfer (of the HTRF type). They arealso of great advantage in more conventional techniques of detection byfluorescence, where the number of fluorophores per carrier molecule, thequantum yield and the molar extinction coefficient of the fluorophoreare predominant criteria for the sensitivity of these systems.

The invention therefore also relates to the use of a fluorescent entityor of a fluorescent conjugate as defined above, as fluorescenttracer(s), for example for detecting and/or determining, byfluorescence, an analyte in a medium liable to contain it or fordetermining an interaction between biomolecules; or for determining abiological activity such as: an enzyme activity, the activation of amembrane-bound receptor, the transcription of a gene, a membranetransport or a variation in membrane polarization, in particular in amethod for screening medicinal products.

The fluorescent conjugates according to the invention can be used asacceptor fluorescent compounds in the presence of donor fluorescentcompounds or as donor fluorescent compounds in the presence of acceptorfluorescent compounds, in particular in fluorescence microscopy, in flowcytometry, in fluorescence polarization or in fluorescence correlation.

They can also be advantageously used as a contrast agent for opticalimaging in vivo.

A subject of the invention is also a method for decreasing thephenomenon of aggregation at the surface of a carrier molecule attachedto a fluorophore, characterized in that a fluorescent entity as definedabove is used in place of said fluorophore.

Finally, a subject of the invention is a method for increasing thequantum yield of a fluorophore attached to a carrier molecule,characterized in that a fluorescent entity as defined above is used as afluorophore.

The fluorescent entities according to the invention can be prepared asdescribed below, by coupling “functionalized” oligonucleotides with afluorophore.

In the present description, the term “functionalized oligonucleotide” isintended to mean an oligonucleotide comprising at least one chemicallyreactive function or a chemical group (such as a fluorescent group)which is not present in a natural oligonucleotide and which results fromthe incorporation of a modified nucleotide or of a non-nucleotide unitcarrying this chemically reactive function or this chemical group. Thischemically reactive function makes it possible, inter alia, to performthe synthesis of conjugates of oligonucleotides or of modifiedoligonucleotides. The terms “chemically reactive function”, “modifiednucleotide”, “non-nucleotide unit” and “oligonucleotide conjugates” areunderstood to be in the sense described, for example, in the review byJ. Goodchild [Conjugates of oligonucleotides and modifiedoligonucleotides: A review of their synthesis and properties.Bioconjugate chemistry, (1990) 1(3), 77-99]. The term “naturaloligonucleotide” denotes a polynucleotide formed by the series ofnucleotide units existing in nucleic acids [Abbreviations and symbolsfor the description of conformations of polynucleotide chains. Eur. J.Biochem. (1983) 131, 9-15].

The following examples illustrate the invention in a nonlimiting manner.

The following abbreviations are used:

-   DTT: dithiothreitol-   DSS: N-(disuccinimidyl) suberate-   GST: glutathione S-transferase-   MOPS: 3-[N-morpholino]propanesulfonic acid-   SPDP: N-succinimidyl-3-(2-pyridyldithio)propionate-   SSMCC: 3-sulfo-N-hydroxysuccinimide ester of    4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-   TEAB: triethylammonium bicarbonate-   TEA Ac: triethylammonium acetate containing 10% of acetonitrile.    I/Synthesis of Conjugates

EXAMPLE 1 Synthesis of the Compounds CY5-T15-hexylamine,CY5-T10-hexylamine and CY5-T5-hexylamine

This example describes the synthesis of an oligonucleotide of sequenceT₅, T10 or T₁₅ functionalized at its 5′ end with a cyanin molecule suchas CY5 which is nonsulfonated, and at its 3′ end with an arm carrying anamine group which can be used to label a biological molecule ofinterest. The general structure of the compound CY15-T10 hexylamine forexample can be symbolized by ^(5′)(CY5-TTT TTT TTT T-hexylamine)₃.

The term “hexylamine” denotes an arm composed of 6 carbon atoms,possibly substituted and carrying an amine function.

In the present example, a 2-hydroxymethyl-6-aminohexanol arm is linkedvia a phosphate bridge formed between the hydroxyl in the 3′ position ofthe nucleotide located at the 3′ end and the 2-hydroxymethyl of the arm.

A solid support of the CPG (controlled pore glass) type conventionallyused for synthesizing oligonucleotides is used. Such a support isreferred to as “functionalized” since grafted onto the CPG is a chemicalstructure carrying a protected amine function capable, after finaldeprotection of the oligonucleotide, of releasing an aliphatic primaryamine function.

A commercially available phosphoramidite derivative of thymidine is usedfor synthesizing the sequence T₁₅.

A commercially available phosphoramidite derivative of a nonsulfonatedcyanin, which makes it possible to directly introduce the fluorescentmarker such as cyanin (CY5) in the 5′ position of the oligonucleotide,is used.

The synthesis is carried out using an automatic DNA synthesizer (AppliedBiosystems type 392) according to the manufacturer's protocol. Thecolumn containing the solid support (CPG) grafted (1 μmol) with a2-O-dimethoxytrityl-6-fluorenylmethoxycarbonylaminohexane-1-succinoyl-longchain alkylamino-CPG derivative is placed on the synthesizer, thesequence T₁₅ is synthesized by performing fifteen cycles of synthesisusing the phosphoramidite derivative of thymidine, and then a couplingcycle is carried out using the phosphoramidite derivative of thenonsulfonated cyanin (CY5).

At the end of this synthesis, the column is subjected to ammoniacaltreatment (approximately 2 ml of 28% aqueous ammonia), making itpossible to cleave the bond between the oligonucleotide and the CPGsupport, according to the manufacturer's protocol. The flask forcollecting the released oligonucleotide is sealed, kept at 50-55° C. for2 h, and then brought back to ambient temperature. The content of theflask (2 ml) is then transferred into a 5 ml polypropylene tube and thenevaporated to dryness under vacuum using a speed-vac. The residue isthen taken up with 500 μl of 10 mM TEAB. The solution obtained containsthe “crude” oligonucleotide and predominantly the desired compound 5(CY5-(T)₁₅-2-oxymethyl-6-aminohexanol)_(3′.)

The compound ^(5′)(CY5-(T)₁₅-2-oxymethyl-6-aminohexanol)₃, is obtainedafter HPLC purification on a LiChrospher RP-18^(e) 250-10 (10μ) column(Merck) using a gradient of acetonitrile in aqueous TEAAc (buffer A: 5%acetonitrile in 25 mM TEAAc, buffer B: 50% acetonitrile in 25 mM TEAAc;flow rate 5 ml/min, linear gradient of 10% B to 20% B in 20 min and alinear gradient of 20% to 100% of B in 10 min. The sequences which areincomplete and which do not comprise CY5 are eluted around 20 min, thefractions containing the desired sequence^(5′)(CY5-(T)₁₅-2-oxymethyl-6-aminohexanol)₃, are collected around 28min (these fractions are blue in color due to the presence of the CY5group).

The fractions containing the desired sequence are pooled, and evaporatedto dryness using a speed-vac, the residue being taken up with purewater. A UV/visible spectrum (210 nm to 750 nm) effected on a dilutionof this solution makes it possible to determine the concentration of theoligonucleotide by its absorbence at 260 nm and to characterize thepresence of the cyanin group (CY5) by its absorbence at 650 nm.

The compounds CY5-T10-hexylamine and CY5-T5-hexylamine are synthesizedin the same way, by varying the number of cycles of synthesis in theautomatic synthesizer.

EXAMPLE 2 Activation of the Compounds CY5-T15-hexyl-amine,CY5-T10-hexylamine and CY5-T5-hexylamine in the 3′ Position with SSMCC

The compound CY5-T15-hexylamine obtained in example 1 is dissolved in a200 mM PO₄ buffer and the pH is adjusted to 8.

250 equivalents of SSMCC are added to the solution obtained. Thereaction mixture is incubated for 30 min at ambient temperature, withstirring.

The compound CY5-T15-hexylamine activated with SSMCC, hereinafterreferred to as CY5-T15-maleimide (hereinafter named CY5-T15 (mal)), ispurified on an HR 10/30 G25 (SF) column in 10 mM PO₄ buffer containing 2mM EDTA, pH 7. The purification is carried out at 60 ml/h.

The solution of CY5-T15-maleimide is concentrated by evaporation in thespeed-vac.

The same procedure is carried out for CY5-T10-hexylamine.

For the activation of the compound CY5-T5-hexylamine, only 150equivalents of SSMCC are used. An additional purification step on an HR10/30 column is necessary.

EXAMPLE 3 Activation of CY5-T15-hexylamine in the 3′ Position withDSS(N-(disuccinimidyl suberate)

The compound CY5-T15-hexylamine obtained in example 1 is taken up in 10μl of 100 mM MOPS buffer, pH 7.6, and 5 μl of acetonitrile.

150 equivalents of DSS (35 mg/ml in DMF) are added to the solutionobtained.

The reaction mixture is incubated overnight at 4° C. with stirring. Thecompound CY5-T15-hexylamine activated with DSS, hereinafter referred toas CY5-T15-NHS, is purified on a NAP 5 G25 (SF) column in 5 mM MOPSbuffer, pH 6.5.

The compound CY5-T15-NHS is then concentrated by several precipitationswith butanol and centrifugations. The pellet is taken up in water.

The same procedure is carried out for the activation of the compoundsCY5-T10-hexylamine and CY5-T5-hexylamine.

EXAMPLE 4 Preparation of the Conjugate CY5-(T)_(n)-hexyl-amine Activatedwith SSMCC (Example 2)—anti-GST Antibody (GSS11)

GSS11-T15-CY5 Batch 02B (mal):

The antibody GSS11 (CIS bio international, France) in 0.1 M carbonatebuffer, pH 9, is activated by adding 8 equivalents of SPDP for 30 min atambient temperature, with stirring, and then adding a finalconcentration of 20 mM of DTT for 15 min at ambient temperature, withoutstirring.

It is purified on an HR 10/10 G25 (SF) column in 0.1 M PO₄ buffer, pH 7.

The antibody thus activated is mixed with the oligonucleotideCY5-T15-maleimide obtained in example 2, with an initialCY5-T15-maleimide/antibody GSS11 molar ratio of 7.6. The incubation isfrom 18 to 20 h at +4° C.

The concentration of the antibody during the coupling is 0.5 mg/ml.

GSS11-T15-CY5 batch 03 (mal):

The antibody GSS11 is activated only by adding DTT at a concentration of5 mM. The same procedure as for batch O₂B above is then carried out,with an initial CY5-T15-maleimide/antibody GSS11 molar ratio of 10.8.

The concentration of the antibody during the coupling is, in this case,0.68 mg/ml.

GSS11-T5-CY5 Batch 01 (mal):

The same procedure as for batch O₂B above is carried out, with aninitial CY5-T5-maleimide/antibody GSS11 molar ratio of 8.

The concentration of the antibody during the coupling is −0.9 mg/ml.

GSS1-T10-CY5 Batch 01 (mal):

The same procedure as for the GSS11-T5-CY5 batch 01 (mal) is carriedout.

The coupling products are purified on an HR 10/30 Superdex 200 column at60 ml/h.

The elution buffer is a 0.1 M phosphate buffer, pH 7.

The purifications are followed using a diode array detector (followed atvarious wavelengths).

EXAMPLE 5 Preparation of the Conjugate CY5-(T)_(n)-hexylamine Activatedwith DSS (Example 3)—anti-GST Antibody (GSS11)

GSS11-T15-CY5 Batch 01 (NHS):

The antibody GSS11 in 0.1 M carbonate buffer, pH 9, is mixed with theCY5-T15-NHS solution obtained in example 3. The initialCY5-T15-NHS/antibody molar ratio is 8. The concentration of the antibodyduring the coupling is 3.3 mg/ml. The incubation is for 30 min atambient temperature without stirring.

GSS11-T15-CY5 Batch 02 (NHS):

The protocol is the same as for GSS11-T15-CY5 batch 01 NHS, with aninitial CY5-T15-NHS/antibody molar ratio of 12. The incubation is for 2h 30 at ambient temperature, but with stirring.

The coupling products are purified on an HR 10/30 Superdex 200 column at60 ml/h. The elution buffer is a 0.1 M phosphate buffer, pH 7.

The purifications are followed using a diode array detector (followed atvarious wavelengths).

EXAMPLE 6 Synthesis of the Conjugate CY5sulfo-GSS11

These conjugates, which do not comprise any oligonucleotide, serve asreference compounds to show the advantages of the conjugates accordingto the invention.

A sulfonated cyanin is used here, and not a nonsulfonated cyanin as inthe previous examples, since the quantum yield of the latter isvirtually zero if it is not coupled to an oligonucleotide.

GSS11-CY5sulfo Batch M5:

Sulfonated cyanin (CY5sulfo) mono NHS is added to antibody GSS11 in 0.1M carbonate buffer, pH 9.

The initial CY5sulfo/antibody molar ratio is 4.

The incubation is for 1 h at ambient temperature with stirring, theconcentration of the antibody during the coupling is 5.2 mg/ml.

The purification is carried out on an HR10/10 G25 (SF) column in 0.1 Mphosphate buffer, pH 7.

GSS11—CY5sulfo Batch M6:

The protocol is the same as for batch M5 above, but with an initialCY5sulfo/antibody molar ratio of 10.

GSS11—CY5sulfo Batch M7:

The protocol is the same as for batch M5 above, but with an initialCY5sulfo/antibody molar ratio of 20.

EXAMPLE 7 Preparation of the Conjugate CY5-A15-cAMP

The compound Cy5-A15-hexylamine is obtained according to a proceduresimilar to that described for the Cy5-T15-hexylamine in example 1, usinga phosphoramidite derivative of adenosine in place of thephosphoramidite derivative of thymidine.

The synthesis of the N-hydroxysuccinimide ester of2′-monosuccinyladenosine 3′,5′-cyclic monophosphate (cAMP-succ-NHS) iscarried out as follows:

2 mg (3.9 μmol) of 2′-monosuccinyladenosine 3′,5′-cyclic monophosphate(sodium salt, Sigma # M9631) are suspended in 15 μl of a solution ofN-hydroxysuccinimide (13.3 mg/ml in anhydrous DMSO), in an eppendorfftube.

35 μl of a solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (200 mg/ml in anhydrous DMSO) are then added to thereaction volume and then placed on a shaker at ambient temperature.

After reaction for 90 minutes, the reaction mixture is diluted with DMSOqs 160 μl, so as to obtain a solution of [cAMP-succ-NHS]=23 nmol/μl,which is used for the synthesis of conjugates.

The cAMP-succ-NHS taken up in H₂O is then mixed with a solution ofCY5-A15-hexylamine in 10 mM TEAB buffer, pH=8.5. During the couplingreaction, the molar ratio is 20 cAMP-NHS per CY5-A15-hexylamine. ThecAMP concentration during the coupling is 5 μmol/ml. The incubation isfor 2 h 30 at ambient temperature with agitation.

The purification is carried out using desalification on a NapS (G25)column in 0.1 M PO₄ buffer, pH 7.

II—Photophysical Properties

EXAMPLE 8 Comparison of the Quantum Yields and of the OD max/OD 604 nmRatio

The quantum yield reflects the efficiency of the fluorescent entity inreleasing the energy which it receives: the higher it is, the moreefficient the fluorescent entity. These quantum yields are measuredusing a fluorimeter. The excitation is carried out at 600 nm, thefluorescence is measured at 615 to 750 nm. The area of the fluorescencespectra obtained is calculated and used to determine the quantum yields.

The final molar ratio (FMR) expresses the number of fluorophorescovalently coupled to the protein.

The absorption spectra of the fluorescent entities make it possible tocalculate the OD max/OD 604 nm ratio. This ratio reflects a possiblephenomenon of aggregation of the fluorophores, for example CY5, at thesurface of the labeled proteins, for example the antibody GSS11 orstreptavidin.

The quantum yield and the OD max/OD 604 nm ratio are determined forvarious compounds synthesized according to the preceding examples.

8.1/The results given in table 1 below concern the reference conjugates(comprising no oligonucleotide) prepared in example 6 above. TABLE 1Final molar OD_(max)/ Quantum ratio (FMR) OD_(604 nm) yieldCY5sulfo-mono NHS — 3.16 19% GSS11-CY5sulfo batch M5 2.20 2.18 11%GSS11-CY5sulfo batch M6 3.80 1.64 4% GSS11-CY5sulfo batch M7 6.00 1.241%

These results show that the coupling of the sulfonated cyanin CY5sulfowith an antibody GSS11 leads to a decrease in the quantum yield,associated in particular with a phenomenon of aggregation of CY5 at thesurface of the antibody, as shown by the decrease in the ODmax/OD 604 nmratio.

The more the number of CY5 molecules per antibody molecule (FMR)increases, the greater this decrease.

8.2/The results given in table 2 below concern the conjugates preparedin examples 4 and 5. The compounds CY5-T10-hexylamine andCY5-T15-hexylamine used as references are prepared as described inexample 1. TABLE 2 Final molar OD_(max)/ Quantum ratio (FMR) OD_(604 nm)yield CY5-T15-hexylamine — 2.79 25% GSS11-T15-CY5 batch 02B (mal) 4.302.59 19% GSS11-T15-CY5 batch 03 (mal) 1.40 2.59 18% GSS11-T15-CY5 batch01 (NHS) 2.00 2.67 20% GSS11-T15-CY5 batch 02 (NHS) 5.10 2.63 20%CY5-T10-hexylamine — 2.78 26% GSS11-T10-CY5 batch 01 (mal) 4.60 2.38 14%

These results show that, when coupling the antibody GSS11 withfluorescent entities according to the invention, a very small decreasein the quantum yield and in the OD max/OD 604 nm ratio is observed, incomparison to the reference conjugate, for increasing FMRs. This clearlyshows that the fluorescent entities according to the invention exhibitcompletely unexpected properties in terms of decreasing the aggregationat the surface of the labeled protein (here the antibody GSS11).

In addition, the result obtained is independent of the method ofactivation of the compound CY5-T15-hexylamine (DSS or SSMCC).

8.3/The results given in table 3 below concern the product prepared inexample 7. The compound Cy5-A15-hexylamine used as a reference isprepared according to the procedure described for Cy5-T15-hexylamine inexample 1. TABLE 3 OD_(max)/OD₆₀₄ Quantum yield CY5-A15-NH2 2.7   16%CY5-A15-cAMP 2.65 19.5%

The results above show that the quantum yield of fluorescence of thefluorescent entities is slightly increased after coupling to the cyclicAMP. The conjugate thus created exhibits no sign of aggregation and thushas unexpected fluorescence properties.

EXAMPLE 9 Fluorescence Spectrum at Constant Antibody Concentration

In order to compare the level of fluorescence obtained with the variousconjugates, fluorescence emission spectra (intensity of fluorescence asa function of the emission wavelength) were produced with a constantconcentration of antibody (50 M). These spectra are produced on an LS50Bspectrofluorimeter (PerkinElmer), with the settings as follows:excitation wavelength 600 nm, emission wavelength 615 to 750 nm,scanning rate 480 nm/min. Based on these spectra, it is possible todetermine the intensity of fluorescence of the conjugate by calculatingthe area of the spectrum obtained by integration.

FIG. 1 gives the relationship between the FMR of the conjugatesGSS11-CY5sulfo and GSS11-T15-CY5 and their intensity of fluorescence.

The following symbols are used:

-   —●— represents the conjugate GSS11-CY5 sulfo-   —▪— represents the conjugate GSS11-T15-CY5

The graph in FIG. 1 shows that, in the case of the conjugatesGSS11—CY5sulfo, the increase in the FMR of the conjugate leads to adecrease in its overall fluorescence, due to the extreme aggregation ofthe CY5 at the surface of the antibody. On the other hand, in the caseof the conjugates GSS11-T15-CY5, the absence of aggregation of the CY5makes it possible to maintain a high quantum yield and, consequently, toobtain an overall fluorescence which is virtually proportional to thenumber of CY5 per antibody.

III—Use of the Fluorescent Entities According to the Invention in Assaysof the FRET (“Fluorescence Resonance Energy Transfer”) Type

EXAMPLE 10

The fluorescent entities according to the invention can be used insystems of the FRET type well known to those skilled in the art.

In the present example, biotinylated Glutathione S-transferase(GST-biotin) is detected by measuring the fluorescence emitted by anacceptor compound, resulting from an energy transfer between a donorcompound (conjugate europium cryptate-streptavidin (K(Eu)-Sa)) and anacceptor containing a fluorescent entity according to the invention(conjugate GSS11-oligonucleotide-CY5).

Assay Protocol:

The assay is carried out using a fluorimeter (Discovery, Packard), theexcitation wavelength of which is 337 nm. The fluorescence is measuredat 665 and 620 nm.

-   Assay buffer: 50 mM Hepes, pH 7, 0.1% BSA, 400 mM KF.-   Reagents: GST-biotin, 20 nM solution-   Donor: Sa-K(EU) NHS (CIS bio international)-   Acceptor: GSS11-XL665 (CIS bio international) used as reference    -   GSS11-T15-CY5 batch 02B mal (ex. 4) FMR 4.3    -   GSS11-T15-CY5 batch 03mal (ex. 4) FMR 1.4    -   GSS11-T15-CY5 batch 01NHS (ex. 5) FMR 2    -   GSS11-T15-CY5 batch 02NHS (ex. 5) FMR 5.1    -   GSS11-T10-CY5 batch 01mal (ex. 4) FMR 4.6    -   GSS11-T15-CY5 batch 01mal (ex. 4) FMR 8.7

The following mixture is incubated for 20 h at ambient temperature:

-   -   50 μl of GST-biotin at 0-0.31-0.62-1.25-2.5 or 5 nM final        concentration    -   100 μl of acceptor at 2.5 nM final concentration    -   50 μl of donor at 1 nM final concentration

FIG. 2 represents the evolution of the signal (% delta F) as a functionof the evolution of the concentration of the GST-biotin (GST-BIOT in nMfinal concentration).

The following symbols are used:

-   —∘— GSS11-XL-665-   —♦— represents the conjugate GSS11-T15-CY5 batch O₂ NHS-   — — represents the conjugate GSS11-T15-CY5 batch 01 NHS-   —Δ— represents the conjugate GSS11-T15-CY5 batch 02B mal-   —x— represents the conjugate GSS11-T15-CY5 batch 03 mal-   — represents the conjugate GSS11-T10-CY5 batch 01 mal

The graph in FIG. 2 shows the advantage of the fluorescent entitiesaccording to the invention as fluorescent labels in an assay of the FRETtype. Specifically, in all cases, the signal observed using thefluorescent entities according to the invention is greater than or equalto that obtained with the reference acceptor compound (XL).

EXAMPLE 11 Lifetime and Efficiency of Transfer of the Conjugates

The lifetimes and the efficiency of transfer obtained in the assay ofthe FRET type such as that of the preceding example were calculated. Theconjugates tested were prepared as described in examples 4 and 5, theconjugate GSS11-XL665 serving as a reference.

The results are given in table 4 below. TABLE 4 Sa-K batch 14 (NHSgeneric product) Lifetime Lifetime of of the the free K Efficiency ofFRET in ms in ms transfer in % (τ_(FRET)) (τ_(cryptate)) 1 −(τ_(FRET)/τ_(cryptate)) GSS11-T10-CY5 0.16 0.969 83% batch 01malGSS11-T5-CY5 0.21 1.030 80% batch 01mal GSS11-T15-CY5 0.21 1.054 79%batch 03mal GSS11-T5-CY5 0.16 1.001 83% batch 02mal GSS11-T15-CY5 0.170.997 83% batch 01NHS GSS11-T15-CY5 0.13 1.038 87% batch 02NHSGSS11-XL665 0.27 1.033 73%

The results show that the efficiency of energy transfer between thedonor compound and the conjugates according to the invention used asacceptors is significantly higher for the conjugates according to theinvention than for the conjugate XL665 (control).

EXAMPLE 12

The fluorescent entities can be used in FRET competition systems wellknown to those skilled in the art.

In the present example, the presence of cyclic AMP (cAMP) in a sample isdetected by observing the inhibition of the fluorescence energy transferoccurring between a donor compound (conjugate europiumcryptate-anti-cAMP monoclonal antibody) and an acceptor compoundcontaining a fluorescent entity (conjugate Cy5-A15-cAMP).

Assay Protocol:

The assay is carried out using a fluorimeter (Rubystar, BMG), theexcitation wavelength of which is 337 nm. The fluorescence is measuredat 665 nm and 620 nm.

-   Assay buffer: 0.1 M phosphate, pH=7, 0.1% BSA, 400 mM KF.-   Reagents: cAMP, solution at 280 nM-   Donor: conjugate Europium cryptate-anti-cAMP monoclonal antibody    (anti-cAMP-K) (Cis bio international)-   Acceptor:-   conjugate Cy5-A15-cAMP (Cisbio international),-   conjugate cAMP-XL665 used as a reference.

The following mixture is incubated for 20 h at ambient temperature:

-   -   25 μl buffer    -   25 μl cAMP at 0-0.07-0.27-1.09-4.37-17.5 or 70 nM final        concentration    -   25 μl cAMP-XL665 or Cy5-A15-cAMP    -   25 μl anti-cAMP-K

FIG. 3 represents the inhibition of FRET signal (DF/DF max) obtained attwo incubation times (1 h and 20 h) in the presence of increasingamounts of cAMP.

The following symbols are used:

-   --- --- cAMP-XL665 incubation time 1H-   ---∘--- cAMP-XL665 incubation time 20H-   —x— Cy5-T15-cAMP incubation time 1H-   —∇— Cy5-T15-cAMP incubation time 20H

The graph in FIG. 3 shows the advantage of the fluorescent entitiesaccording to the invention as fluorescent labels in an assay of the FRETcompetition type. The sensitivity of the test is improved by using thefluorescent entities according to the invention, whatever the incubationtime of the experiment. In addition, the loss of sensitivity observedwith the reference acceptor (XL665) between 1 h and 20 h of incubationdisappears when the fluorescent entities according to the invention areused.

1. A fluorescent entity comprising a fluorophore, with the exception ofa rare earth metal cryptate, covalently attached to one or moreoligonucleotide(s) or oligonucleotide analog(s), characterized in thatit comprises at least one functional group, introduced or generated onthe fluorophore or one of the oligonucleotides or oligonucleotideanalogs.
 2. The entity as claimed in claim 1, characterized in that theoligonucleotide or the oligonucleotide analog comprises from 2 to 60nucleotide units.
 3. The entity as claimed in claim 1, characterized inthat the functional group can be attached to said entity via a spacerarm.
 4. The entity as claimed in claim 1, characterized in that thefluorophore comprises one or more aromatic rings and has a highmolecular extinction coefficient, greater than 20 000, preferablygreater than 50
 000. 5. The entity as claimed in claim 1, characterizedin that the fluorophore is chosen from rhodamines, cyanins, squaraines,bodipys, fluoresceines and their derivatives.
 6. The entity as claimedin claim 1, characterized in that the functional group is chosen fromthe groups: maleimide, carboxylic acid, haloacetamide, alkyl halide,azido, hydrazido, aldehyde, ketone, amino, sulfhydryl, isothiocyanate,isocyanate, monochlorotriazine, dichlorotriazine, aziridine, sulfonylhalide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimideester, imido ester, hydrazide, azidonitrophenyl, azidophenyl, azide,3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula:

where n ranges from 0 to 8 and p is equal to 0 or 1, and Ar is a 5- or6-membered heterocycle comprising 1 to 3 hetero atoms, optionallysubstituted with a halogen atom.
 7. The fluorescent entity as claimed inclaim 1, of formula (I):

in which: A represents a group chosen from:

—N(R₃)_(r) r=2 or 3 the dashed lines each represent the carbon atomsrequired to form 1 to 3 fused rings, the groups R₃ being attached tothese rings; X and Y each represent N, C═O, O, S or C(CH₃)₂ m has avalue 1, 2, 3 or 4; q has a value 1, 2 or 3; (R₃)_(q) represents qgroups R₃, which may be identical or different; the groups R₁, R₂ and R₃are identical or different and are chosen from hydrogen; a group—(CH₂)_(s)-Z in which s ranges from 0 to 4 and Z represents a group CH₃,SO₃H, OH or N⁺R₁R₂R₃ in which R₁, R₂ and R₃ are as defined above; afunctional group is chosen from the groups: maleimide, carboxylic acid,haloacetamide, alkyl halide, azido, hydrazido, aldehyde, ketone, amino,sulfhydryl, isothiocyanate, isocyanate, monochlorotriazine,dichlorotriazine, aziridine, sulfonyl halide, acid halide,hydroxy-succinimide ester, hydroxysulfosuccinimide ester, imido ester,hydrazide, azidonitrophenyl, azidophenyl, azide,3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula:

where n ranges from 0 to 8 and p is equal to 0 or 1 and Ar is a 5- or6-membered heterocycle comprising 1 to 3 hetero atoms, optionallysubstituted with a halogen atom; and an oligonucleotide oroligonucleotide analog optionally comprising a functional group as said;R₄ is chosen from: H; OH; CH₃; Cl and the groups of formula:

the substituents R₄ in the allylic position possibly forming, with thepolyethylenic chain, 1 to 3 fused rings containing from 4 to 14 atoms,which may or may not be saturated, said rings possibly containing one ormore atoms of O, N and S, and possibly being optionally substituted withan oxo group.
 8. The fluorescent entity as claimed in claim 1, offormula (II) or (III):

or in which the dashed lines each represent the carbon atoms required toform 1 to 3 fused rings, the groups R₃ being attached to these rings; Xrepresents N, C═O, O, S or C(CH₃)₂; m has a value 1, 2, 3 or 4; q has avalue 1, 2 or 3; (R₃)₂ represents q groups R₃, which may be identical ordifferent; the groups R₁ and R₃, which may be identical or different,are chosen from hydrogen; a group —(CH₂)_(s)-Z in which s ranges from 0to 4 and Z represents a group CH₃, SO₃H, OH or N⁺R₁R₂R₃ in which R₁, R₂and R₃ are as defined above; a functional group is chosen from thegroups: maleimide, carboxylic acid, haloacetamide, alkyl halide, azido,hydrazido, aldehyde, ketone, amino, sulfhydryl, isothiocyanate,isocyanate, monochlorotriazine, dichlorotriazine, aziridine, sulfonylhalide, acid halide, hydroxy-succinimide ester, hydroxysulfosuccinimideester, imido ester, hydrazide, azidonitrophenyl, azidophenyl, azide,3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula:

where n ranges from 0 to 8 and p is equal to 0 or 1, and Ar is a 5- or6-membered heterocycle comprising 1 to 3 hetero atoms, optionallysubstituted with a halogen atom; an oligonucleotide or oligonucleotideanalog optionally comprising a functional group as said; R₄ is chosenfrom: H; OH; CH₃; Cl and the groups of formula:

the substituents R₄ in the allylic position possibly forming, with thepolyethylenic chain, 1 to 3 fused rings containing from 4 to 14 atoms,which may or may not be saturated, said rings possibly containing one ormore atoms of O, N and S, and possibly being optionally substituted withan oxo group.
 9. The fluorescent entity as claimed in claim 1, offormula (IV), (V), (VI) or (VII):

in which R₁, R₂, R₃ and R₅ are identical or different and are chosenfrom hydrogen; a group —(CH₂)_(s)-Z in which s ranges from 0 to 4 and Zrepresents a group CH₃, SO₃H, OH or N⁺R₁R₂R₃ in which R₁, R₂ and R₃ areas defined above; a functional group is chosen from the groups:maleimide, carboxylic acid, haloacetamide, alkyl halide, azido,hydrazido, aldehyde, ketone, amino, sulfhydryl, isothiocyanate,isocyanate, monochlorotriazine, dichlorotriazine, aziridine, sulfonylhalide, acid halide, hydroxy-succinimide ester, hydroxysulfosuccinimideester, imido ester, hydrazide, azidonitrophenyl, azidophenyl, azide,3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula:

where n ranges from 0 to 8 and p is equal to 0 or 1, and Ar is a 5- or6-membered heterocycle comprising 1 to 3 hetero atoms, optionallysubstituted with a halogen atom; and an oligonucleotide oroligonucleotide analog optionally comprising a functional group as said;q has a value 1, 2 or
 3. 10. The fluorescent entity as claimed in claim1, of formula (VIII):

in which the substituents R₆ to R₁₂ are chosen from: hydrogen; ahalogen; an alkyl; a cycloalkyl; aryl; arylalkyl; acyl; sulfo; afunctional group is chosen from the groups: maleimide, carboxylic acid,haloacetamide, alkyl halide, azido, hydrazido, aldehyde, ketone, amino,sulfhydryl, isothiocyanate, isocyanate, monochlorotriazine,dichlorotriazine, aziridine, sulfonyl halide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,hydrazide, azidonitrophenyl, azidophenyl, azide,3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula:

where n ranges from 0 to 8 and p is equal to 0 or 1 and Ar is a 5- or6-membered heterocycle comprising 1 to 3 hetero atoms, optionallysubstituted with a halogen atom; and an oligonucleotide oroligonucleotide analog optionally comprising a functional group chosenfrom those mentioned as said.
 11. The entity as claimed in claim 7, offormula (IX):

in which R₁, R₂, R₃, R₄, X, Y, m and q are as defined above.
 12. Theentity as claimed in claim 11, in which X and Y each represent a groupC(CH₃)₂.
 13. The entity as claimed in claim 11, in which R₁ and R₂represent an alkyl comprising from 1 to 4 carbon atoms or a group offormula below, at least one of the groups R₁ and R₂ representing a groupof formula below:

R₄ represents hydrogen q=1, m=2 R₃ represents hydrogen; a groupCH₂)_(s)-Z in which s ranges from 0 to 4 and Z represents a group CH₃,SO₃H, OH or N⁺R₁R₂R₃ in which R₁, R₂ and R₃ are as defined above; afunctional group is chosen from the groups: maleimide, carboxylic acid,haloacetamide, alkyl halide, azido, hydrazido, aldehyde, ketone, amino,sulfhydryl, isothiocyanate, isocyanate, monochlorotriazine,dichlorotriazine, aziridine, sulfonyl halide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,hydrazide, azidonitrophenyl, azidophenyl, azide,3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula:

where n ranges from 0 to 8 and p is equal to 0 or 1, and Ar is a 5- or6-membered heterocycle comprising 1 to 3 hetero atoms, optionallysubstituted with a halogen atom; an oligonucleotide or oligonucleotideanalog optionally comprising a functional group as said; R₄ is chosenfrom: H; OH; CH₃; Cl and the groups of formula:

the substituents R₄ in the allylic position possibly forming, with thepolyethylenic chain, 1 to 3 fused rings containing from 4 to 14 atoms,which may or may not be saturated, said rings possibly containing one ormore atoms of O, N and S, and possibly being optionally substituted withan oxo group.
 14. The entity as claimed in claim 1, characterized inthat the fluorophore is covalently attached to the oligonucleotideeither directly or via a spacer arm.
 15. The entity as claimed in claim14, characterized in that the fluorophore is attached to theoligonucleotide via a spacer arm consisting of a divalent organicradical chosen from linear or branched C₁-C₂₀ alkylene groups optionallycontaining one or more double bonds or triple bonds and/or optionallycontaining one or more hetero atoms, such as oxygen, nitrogen, sulfur,phosphorus, or one or more carbamoyl or carboxamido group(s); C₅-C₈cycloalkylene groups and C₆-C₁₄ arylene groups, said alkylene,cycloalkylene or arylene groups optionally being substituted with alkyl,aryl or sulfonate groups.
 16. The entity as claimed in claim 15,characterized in that the spacer arm is chosen from the groups:

in which n₁ and n₂ are between 2 and
 6. 17. The entity as claimed inclaim 1, characterized in that the oligonucleotide comprises from 5 to60, in particular 5 to 20, preferably from 5 to 15, nucleotide units.18. The entity as claimed in claim 17, characterized in that theoligonucleotide consists of a series of ribonucleotide ordeoxyribonucleotide units attached to one another via bonds of thephosphodiester type.
 19. The entity as claimed in claim 17,characterized in that the oligonucleotide consists of a series ofribonucleotide or deoxyribonucleotide units or of nucleotide analogunits modified on the sugar or on the base, attached to one another bynatural internucleotide bonds of the phosphodiester type, some of theinternucleotide bonds being optionally replaced with phosphonate,phosphoramide or phosphorothioate bonds.
 20. The entity as claimed inclaim 17, characterized in that the oligonucleotide consists of a seriescomprising both ribonucleotide or deoxyribonucleotide units attached toone another by bonds of the phosphodiester type and nucleoside analogunits attached to one another by amide bonds.
 21. The entity as claimedin claim 17, characterized in that the oligonucleotide consists of aseries of ribonucleotide or deoxyribonucleotide units attached to oneanother by bonds of the phosphodiester type and of nucleoside analogunits attached to one another by amide bonds, said oligonucleotidecomprising at least 5 internucleotide bonds of the phosphodiester typeat the end intended to be attached to the fluorophore.
 22. The entity asclaimed in claim 1, characterized in that the functional group is anamine function of a nucleotide unit of the oligonucleotide or of theoligonucleotide analog, or results from the reaction of a free aminefunction of a nucleotide unit of the oligonucleotide or theoligonucleotide analog, with a group chosen from the groups: ester,carboxylic acid, isothiocyanate, aldehyde, carbonyl, sulfonyl halide,alkyl halide, azide, hydrazide, dichlorotriazine, anhydride,haloacetamide, maleimide and sulfhydryl.
 23. The entity as claimed inclaim 1, characterized in that the functional group results from thereaction of a free amine function of a nucleotide unit of theoligonucleotide or of the oligonucleotide analog, with anN-hydroxysuccinimidyl ester.
 24. The entity as claimed in claim 1,characterized in that the functional group(s) is (are) attached to thefluorophore and/or to the oligonucleotide by a spacer arm consisting ofa divalent organic radical, chosen from linear or branched C₁-C₂₀alkylene groups optionally containing one or more double bonds or triplebonds and/or optionally containing one or more hetero atoms, such asoxygen, nitrogen, sulfur, phosphorus, or one or more carbamoyl orcarboxamido group(s); C₅-C₈ cycloalkylene groups and C₆-C₁₄ arylenegroups, said alkylene, cycloalkylene or arylene groups being optionallysubstituted with alkyl, aryl or sulfonate groups.
 25. The entity asclaimed in claim 24, characterized in that the spacer arm is chosen fromthe groups:

in which n, and n₂ are between 2 and
 6. 26. A fluorescent conjugateconsisting of an entity as claimed in claim 1 covalently attached to acarrier molecule.
 27. The conjugate as claimed in claim 26,characterized in that the fluorophore of the fluorescent entity iscyanin-5, the oligonucleotide of said entity has the sequence A₁₅ andthe carrier molecule is cAMP.
 28. The conjugate as claimed in claim 26,characterized in that the final molar ratio is greater than 0 and lessthan 100, preferably greater than 0 and less than
 20. 29. Thefluorescent conjugate as claimed in claim 26, characterized in that thecarrier molecule is an antibody, an antigen, an intracellular messenger,an intercellular messenger, a protein, a peptide, a hapten, a lectin,biotin, avidin, streptavidin, a toxin, a carbohydrate, anoligosaccharide, a polysaccharide, a nucleic acid, a hormone, a vitamin,a medicinal product, a polymer, a polymeric particle, glass, a particleof glass or a surface made of glass or of a polymer.
 30. The fluorescentconjugate as claimed in claim 29, characterized in that the carriermolecule is an antibody or streptavidin.
 31. The use of a fluorescententity as claimed in claim 1, as a fluorescent tracer.
 32. The use asclaimed in claim 31, for detecting and/or determining, by fluorescence,an analyte in a medium liable to contain it.
 33. The use as claimed inclaim 32, for determining an interaction between biomolecules; or fordetermining a biological activity such as: an enzyme activity, theactivation of a membrane-bound receptor, the transcription of a gene, amembrane transport or a variation in membrane polarization.
 34. The useas claimed in claim 31, in a method for screening medicinal products.35. The use as claimed in claim 34, in which a fluorescent conjugate isused as an acceptor fluorescent compound in the presence of a donorfluorescent compound.
 36. The use as claimed in claim 35, in which afluorescent conjugate is used as a donor fluorescent compound in thepresence of an acceptor fluorescent compound.
 37. The use as claimed inclaim 35, in fluorescence microscopy, in flow cytometry, in fluorescencepolarization or in fluorescence correlation.
 38. The use of a conjugateas claimed in claim 26, as a contrast agent for optical imaging in vivo.39. A method for increasing the fluorescence intensity of a fluorophoreattached to a carrier molecule, characterized in that a fluorescententity as claimed in claim 1 is used as a fluorophore.
 40. A method fordecreasing the phenomenon of aggregation at the surface of a carriermolecule attached to a fluorophore, characterized in that a fluorescententity as claimed in claim 1 is used in place of said fluorophore.
 41. Amethod for increasing the quantum yield of a fluorophore attached to acarrier molecule, characterized in that a fluorescent entity as claimedin claim 1 is used as a fluorophore.